Different cyclic mechanical loading patterns alter expression of osteogenic markers in a fibroblast-osteoblast co-culture model resembling the tendon-to-bone interface after autologous ACL reconstruction

Abstract

Objectives: The aim of the study was to evaluate the influence of different cyclical mechanical loading patterns on co-cultures of fibroblasts and osteoblasts in vitro, simulating the conditions of the tendon-to-bone interface after anterior cruciate ligament reconstruction. Methods: Tendon-derived rodent fibroblasts (TDF) and osteoblast-like cells (OBL) were co-cultured to simulate the tendon-to-bone interface. Cyclical loading was applied for one hour twice a day for three days, with a frequency of 1 Hz and 3 % strain. Alkaline phosphatase (AP), osteocalcin (OC), collagen type 1 (COL1A1), and bone morphogenetic protein 2 (BMP-2) gene expression and protein deposition were detected by real-time polymerase chain reaction (qPCR) and immunocytochemical analysis. Results: Mechanical loading significantly decreased AP, OC, and COL1A1 gene expression in both OBL and TDF, compared to non-loaded cultures. However, mechanical load increased gene expression of the same marker genes including BMP-2 during co-culture. Immunocytochemistry demonstrated increased deposition of corresponding proteins in the same range, independent of culture conditions. Higher depositions of BMP-2 were shown under loading conditions for osteoblast and TDF monocultures. Prolongation of mechanical loading resulted in cell detachment and spheroid formation. Conclusion: Cyclical mechanical loading caused downregulation of genes involved in osteointegration and osteoinduction, such as OC, ALP, and COL1A1 in monocultures of osteoblasts and fibroblasts; co-cultures lacked this phenomenon. Immunocytochemistry and qPCR analysis showed slight upregulations of marker genes and corresponding proteins. This might be due to the potential stabilising effects of osteoblast-fibroblast cross talk in the co-culture environment, resembling fibrocartilage formation at the tendon-to-bone interface.